The herpes simplex virus type 1 (HSV-1) UL25 protein (pUL25) is a minor capsid protein that is essential for packaging the full-length viral genome into preformed precursor capsid. It is also important in virus entry and recently has been implicated in the egress of the virus from the cell (Coller et al., 2007, Preston et al., 2008). The crystallographic structure of an N-terminally truncated form of pUL25 (residues 134-580) has been determined to 2.1 Å, revealing a protein with a novel fold that consists mostly of a-helices and a few minor b-sheets (Bowman et al., 2006). An unusual feature of the protein is the presence of numerous flexible loops extending out from the stable core and its distinctive electrostatic distribution. Five of the extended loops contain unstructured regions, L1-L5, with three additional unstructured amino acids, L6, located at the carboxyl terminus of the protein (Bowman et al., 2006). Four potentially functional clusters of residues, C1-C4, were identified on the surface of the protein using evolutionary trace analysis (Lichtartge & Sowa, 2002). To examine the function of the protein in relation to its structure, site-directed mutations were engineered into the UL25 gene in a protein expression plasmid. A series of mutant proteins was generated, each protein containing a deletion of the unstructured residues in one of the six regions, L1-L6. Another set of mutant proteins were constructed with each member containing substitutions of selected amino acids within one of the four potentially functional clusters, C1-C4, or substitutions of the three disordered amino acids in L6. The amino acid substitutions were generally to alanine, but in one case where the SIFT program predicted alanine would not affect the function of the protein an alternative residue was substituted. To determine the functional significance of the uncrystallised part of pUL25, residues 1-133, three deletion mutant proteins that spanned this region (pUL25D1-45, pUL25D1-59 and pUL25D1-133) were included in the study. Although an existing UL25 null mutant, KUL25NS, was available at the beginning of the project for analysis of the mutant proteins, it had been made by the insertion of multiple stop codons in the UL25 ORF and as a result some UL25 sequences were still present within the virus genome. Consequently, during complementation assays recombination between the UL25 sequences in the KUL25NS genome and the transfected expression plasmid generated low levels of wild-type (wt) progeny virus. To improve the sensitivity of the assay, a new deletion mutant, ΔUL25MO, was created that lacked the entire UL25 gene. This mutant failed to form plaques in non-permissive Vero cells and grew well in the complementing cell line, 8-1. However, contrary to previously published work, electron microscopic (EM) analysis revealed that DNA-containing capsids as well as A- and B-capsids were present in the nuclei of both ΔUL25MO- and KUL25NS-infected cells. As expected, none of the progeny from ΔUL25MO-infected Vero cells expressing the wt pUL25 formed plaques on non-permissive cells. Of the 17 mutant UL25 proteins screened in the complementation assay, nine failed to complement the growth of ΔUL25MO in Vero cells. Three of the non-complementing mutant proteins examined altered the phenotype of ΔUL25MO in a transient DNA packaging assay, allowing the mutant virus to package full-length genomes in U2OS cells co-infected with ΔUL25MO and a mammalian baculovirus vector containing the mutant UL25 gene. These results indicate that viral assembly was disrupted in these cells following DNA packaging. However, five of the mutant proteins did not change the pattern of DNA encapsidation of ΔUL25MO in this system, suggesting that the wild-type residues mutated in these proteins are critical for packaging virus DNA. To determine at which point in the virus growth cycle the post-packaging blocks occurred, EM was used to investigate the pattern of virus assembly in ΔUL25MO-infected cells expressing either of the three packaging-competent mutant proteins. In addition, fluorescent in-situ hybridisation (FISH) analysis was performed to establish the distribution of virus DNA in these cells. The results showed that in ΔUL25MO-infected cells expressing two of the mutant pUL25s the C-capsids failed to exit the nucleus, whereas in cells expressing the third post-packaging mutant protein C-capsids were seen in both the nucleus and the cytoplasm. The FISH data confirmed the EM observations. These studies show that two regions of pUL25 are important for egress of the C-capsids from the nuclei. Since these two regions lie in close proximity to each other on the surface of the molecule they may represent a single functional interface of the protein. In addition, another region of pUL25 was identified that was essential for the interactions required for virus assembly after the C-capsids are released into the cytoplasm. The 62 carboxyl-terminal region of the UL36 gene product (pUL36) has previously been shown to contain a capsid-binding domain (CBD) that interacts with pUL25 (Coller et al., 2007). A GST-pull down assay was used to determine whether the mutations in the post-packaging mutant proteins disrupted the interaction of pUL25 with the CBD of pUL36. However, all of these mutant proteins and the wt pUL25 bound to the pUL36 CBD GST fusion protein. In summary, three different classes of pUL25 mutants, each of which affect a different essential function of pUL25, have been identified, revealing that pUL25 is indeed a versatile viral protein. These mutants provide the first evidence that this DNA packaging protein is crucial for virus assembly at two different stages after DNA encapsidation, one in nuclear egress of C-capsids and the other in the assembly of the virus in the cytoplasm.

No description available.

Bluetongue (BT) is a non-contagious arthropod transmitted virus that infects ruminants including sheep, cattle and goats. Bluetongue virus (BTV) is economically important due to the severity of disease in naïve animals and the financial cost of the development and implementation of effective vaccines and diagnostic tests, and restrictions on animal movements and trade. Bluetongue virus encapsidates the viral genome, composed of ten linear dsRNA genome-segments, encoding seven structural protein (VP1-VP7) and five non-structural protein (NS1-NS5). The largest-outer-coat and cell-attachment protein of bluetongue virus (BTV), is VP2 (encoded by genome segment-2 (Seg-2)), which elicits protective neutralising antibody (nAb) and total binding antibody (bAb) responses in infected animals. VP2 is the least conserved of the BTV proteins and the nAbs, which are primarily BTV serotype-specific, can be used to identify different serotypes in neutralisation-assays. To-date 27 BTV serotypes have been recognised, with several other putative serotypes currently under investigation. This study investigates the complexity of antigenic cross-reactions between BTV strains belonging to different serotypes, by neutralising and/or total binding antibodies targeting VP2. VP2s from multiple BTV serotypes were expressed using a novel, plant-based, transient- expression system, in Nicotiana benthamiana. The expressed proteins were purified then used to inoculate IFNAR (-/-) mice, using a homologous prime boost vaccination strategy. The purified proteins were also used to immunise rabbits to obtain polyclonal antisera. Sheep polyclonal reference antisera, raised against reference strains of the different BTV serotypes, were also used in this study. All of these antisera were tested for cross-reactivity in serum neutralisation tests and by indirect ELISA. The antigenic relationships observed were compared and quantified using antigenic cartography. The antigenic data was then compared to the VP2 amino acid phylogenetic sequence data. The VP2 proteins were shown to raise nAbs and elicit a protective immune response in IFNAR (-/-) mice. A broad range of antigenic cross-reactivities were detected using the two panels of antisera, identifying serological relationships between different serotypes. These cross-reactivities showed similarities but were not identical to the phylogenetic relationships exhibited by VP2. The evaluation of recombinant VP2s and observed antigenic differences/relationships have important implications for disease surveillance and the possibility of developing serological, type-specific, diagnostic tests and cross-reactive vaccines.

Intrinsic and innate immunity play pivotal roles in limiting the replication of invading viral pathogens. Intrinsic immunity is constitutive and mediated by pre-existing host cell restriction factors (e.g., promyelocytic leukemia-nuclear body (PML-NB) constituent proteins) which directly confer antiviral properties. On the other hand, innate immunity is inducible and upregulated in response to infection. Pattern recognition receptors (PRRs) (e.g., interferon gamma inducible protein 16 (IFI16)) sense pathogen-associated molecular patterns (PAMPs) and induce downstream signaling cascades leading to the induction of Interferon-stimulated gene (ISG) products that confer antiviral properties. These two arms of immunity represent the first line of intracellular defense to HSV-1 infection. Indeed, rapid recruitment of intrinsic and innate immune factors to viral DNA (vDNA) has a significant bearing on the outcome of infection. However, the spatial and temporal regulation of this recruitment remains poorly defined due to the technical challenges associated with vDNA detection at multiplicities of infection (MOI) that do not saturate intrinsic host factors. Utilizing 5-Ethynyl-2’-deoxyuridine (EdU) labeling of HSV-1 DNA in combination with click chemistry, we directly visualized input viral genomes under low MOI conditions (MOI of ≤ 3 PFU/cell) at 30-90 minutes post-addition of virus (mpi). This protocol is sensitive, specific, and compatible with indirect immunofluorescence (IF) staining protocols, providing a valuable assay to investigate the temporal recruitment of immune regulators to infecting vDNA. Upon entry of vDNA into the nucleus, PML-NB associated restriction factors (e.g., PML, SP100, and Daxx) were rapidly recruited to infecting viral genome foci. This process occurred in a PML-dependent manner and led to genome entrapment and silencing within PML-NBs. Interestingly, genome entrapment was observed during both wild-type (WT) and ICP0-null mutant (ΔICP0) HSV-1 infection. During WT HSV-1 infection, ICP0 induced PML degradation and the dispersal of PML-NB restriction factors, highlighting the importance of ICP0 to release viral genomes entrapped within PML-NBs to stimulate the onset of lytic HSV-1 replication. During ΔICP0 HSV-1 infection, vDNA remained stably entrapped within PML-NBs leading to a repression in viral gene expression and a restriction in plaque formation. Importantly, IFI16 was not stably recruited to vDNA entrapped within PML-NBs, and ISG expression was not induced under low MOI conditions that do not saturate PML-NB intrinsic host defenses. These data demonstrate that vDNA entry into the nucleus alone is not sufficient to stimulate the induction of innate immunity. Saturation of intrinsic host defenses under high MOI conditions stimulated the stable recruitment of IFI16 to infecting viral genomes, and induced ISG expression in a PML-, IFI16-, and Janus-associated kinase (JAK)-dependent manner. The induction of this innate immune response was dependent on the onset of vDNA replication, as treatment of the infected cell monolayers with phosphonoacetic acid (PAA), a vDNA polymerase inhibitor, inhibited ISG induction in a dose-dependent manner. Unlike PML depletion, inhibition of JAK signaling failed to relieve the plaque formation defect of ΔICP0 HSV-1, but instead significantly enhanced virus yields. Collectively, these data, for the first time, demonstrate a temporal and sequential induction of intrinsic and innate immunity during HSV-1 infection. Intrinsic immunity is induced within minutes of nuclear infection to restrict the initiation of viral gene transcription and the onset of lytic replication. Escape from this intrinsic repression and initiation of vDNA replication, which takes several hours, triggers the induction of innate immunity. ISG products establish an antiviral state within infected and neighboring uninfected cells to constrict viral propagation and limit the spread of infection. We identify dual roles for PML in the regulation of intrinsic and innate immunity to HSV-1 infection. However, these host defenses are counteracted by the viral ubiquitin ligase ICP0, which targets PML for degradation to promote vDNA release from PML-NBs in order to evade intrinsic viral genome silencing from the onset of nuclear infection.

The horse (Equus caballus) is an economically and scientifically important species of mammal. The horse genome (and that of other mammals) contains thousands of sequences derived from retroviruses, called endogenous retroviruses (ERVs). These sequences are highly informative about the long-term interactions of retroviruses and hosts. They are also interesting because they have influenced the evolution of mammalian genomes in various ways. Horses belong to the family Equidae in the order Perissodactyla - comprising 16 extant species of strict herbivores adapted for running and dietary specialisation. This PhD thesis describes my work developing and applying a novel bioinformatics approach for characterising ERVs. I used this approach to characterise ERVs in genomes of Hippomorpha species in relation to those found in a representative of the Ceratomorpha - the white rhinoceros (Ceratotherium simum). Through comparative analysis of these three genomes, I derive a calibrated timeline describing the process through which ERV diversity has been generated in the equine germline. My project has provided an overview of retrotranspositional activity in perissodactyl ERV lineages and identified individual ERV loci that show evidence of involvement in physiological processes and/or pathological conditions. The dataset generated in this project will be of great utility for future studies aiming to investigate the potential functional roles of equine ERVs and their impact on equine evolution.

BT is endemic in India, particularly in the South and BTV strains belonging to twelve BTV-serotypes (BTV-1, 2, 3, 4, 5, 9, 10, 12, 16, 21, 23 and 24) have been isolated since 2001. A pentavalent inactivated BTV vaccine containing serotypes 1, 2, 10, 16 and 23, is currently being used in the South. This study focusses on the isolation and the typing of BTV strains from Southern India, 2014-15, then generation of full length sequence data, primarily for BTV genome Seg-2 to provide molecular epidemiology information concerning the strains circulating in the region. Suspected field outbreaks of BTV from Telangana, Andhra Pradesh, Karnataka and Tamil Nadu states in Southern India during 2014-2015 were attended to collect well documented blood and necropsy clinical samples from mixed population of small ruminants and cattle. Of the 447 field samples (EDTA blood, spleen, lymph node and saliva), 236 gave positive results for BTV RNA by serogroup-specific real-time RT-PCR targeting Seg-9 and Seg-10 for 2014 and 2015 samples respectively. A total of 141 BTV virus isolations were made from 141 serogroup-specific qRT-PCR positive samples, by infecting KC and BHK cells. The isolated 63 BTVs were serotyped by qRT-PCR assay targeting Seg-2 of the BTV-1 to BTV-24, BTV-26, BTV-27 and BTV-29, confirming the current co-circulation of 7 BTV serotypes, including BTV-1, BTV-2, BTV-4, BTV-5, BTV-9, BTV-10 and BTV-12 in 62 BTV isolates. Dual and triple BTV co-infections with these serotypes were detected in 22 and 4 BTV isolates, respectively. None of the serotypes present in the pentavalent vaccine were identified in any of the 22 BTV isolates from vaccinated animals, suggesting that the vaccine was indeed effective against the homologous serotype field strains. Among the 7 serotypes identified by Seg-2 based ORF sequence analysis of 46 BTV isolates (BTV-1, BTV-2, BTV-4, BTV-5, BTV-9, BTV-10 and BTV-12), 4 serotypes BTV-1, BTV-2, BTV-4 and BTV-9, belonged to eastern topotypes, whereas 3 serotypes, BTV-5, BTV-10 and BTV-12 were identified as western topotypes. For 4 BTV isolates, the serotype detected by qRT-PCR was different of that detected by NGS. The full genome sequencing and analyses is still ongoing in Glasgow. This data will assist in understand the epidemiology of the BT in India and implementation of effective BTV control to ensure the use of appropriate BTV serotype and strains in the available BT vaccine in India.

<p> Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the known human cancer viruses, causing Kaposi's sarcoma and primary effusion lymphoma in immunosuppressed patients. Although of medical concern, the mechanisms through which the virus causes cancer remain poorly understood. Researchers speculate that the lytic phase contributes to the development of human cancers by this virus.</p><p> KSHV, like other herpesviruses, is predominantly latent in the human host, but undergoes lytic activation to produce infectious viral particles. In the process, the virus hijacks the host machinery to express large quantities of viral genes via a process known as the host shutoff effect. The virus then replicates its DNA and assembles viral capsids within nuclear viral replication compartments. Viral proteins act in various locations within the cell depending on their function. However little is known about the location of viral transcripts and how their localization relates to their function. Thus I sought to understand the localization of viral transcripts to gain insight into the spatiotemporal regulation of the lytic phase.</p><p> Using fluorescence in situ hybridization (FISH) and immunofluorescence (IF), I observed that particular viral transcripts accumulate within the nucleus in or near replication compartments. This occurs late in the lytic phase coinciding with viral DNA replication. My findings indicate that the mechanism is independent of the host shutoff effect and splicing, but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, polyadenylated nuclear (PAN) RNA. PAN RNA is essential for the viral life cycle and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.</p><p> One key regulator of the KSHV lifecycle is a long noncoding RNA (IncRNA) called the polyadenylated nuclear (PAN) RNA. PAN RNA is an early gene product comprising nearly 80% of total polyadenylated cellular transcripts in lytic infected cells. Studies on its function demonstrate that PAN RNA is a regulator of virion production through modulation of viral genes.</p><p> A glimpse into the mechanism comes from recent <u>ch</u>romatin isolation by RNA purification (CHIRP) studies on lytic KSHV-infected B lymphocytes. The studies revealed widespread binding of PAN RNA to viral and host chromatin, but could not identify an underlying mechanism. The most feasible approach to study function is a genetic knockout. However, a complete PAN RNA gene deletion is unachievable in the KSHV genome due to an overlapping open reading frame, K7. In a related gammaherpesvirus, rhesus rhadinovirus (RRV), a computational search uncovered a PAN RNA homologue, whose sequence does not overlap with any known genes. I found that RRV PAN RNA is present at about 150,000 copies per cell and organized the purchase of RRV &Delta;PAN RNA constructs to facilitate study of PAN RNA's mechanism.</p><p> Capitalizing on the RRV homolog, Dr. Johanna B. Withers and I compared changes in chromatin association by PAN RNA between homologs and over the lytic phase with CHART (<u>c</u>apture <u>h</u>ybridization <u> a</u>nalysis of <u>R</u>NA targets). After careful analysis, the data suggest that chromatin-association by PAN RNA is nonspecific and that the mechanism of regulation by PAN RNA is not primarily related to chromatin remodeling. With this is mind, I looked to another potential mechanism, one related to binding by nuclear relocalized cytoplasmic polyAbinding protein (PABPC). </p><p> Both PAN RNA homologs associate with several host proteins, one of which is cytoplasmic polyA-binding protein (PABPC). Upon lytic induction, SOX, the host shutoff mediator, facilitates degradation of messages in the cytoplasm, causing the PABPC to relocalize to the nucleus. Nuclear relocalized PABPC binds KSHV PAN RNA at ~8-10 proteins per RNA molecule. I hypothesize that a function of PAN RNA is to act as nuclear relocalized PABPC sponge to facilitate preferential expression of viral genes and assembly of virions.</p><p> I designed mutant to capitalize on a unique feature of PAN RNA, the triple helical stabilization element (ENE). At the 3' end a triple helix (ENE) forms with polyA tail, protecting PAN RNA from deadenylation, stabilizing it. I reduced the length of the polyA-tail to eight adenylates, which permits formation of the triple helix, but falls below the 20-adenylate footprint of PABPC. The RRV PAN constructs would supplement RRV &Delta;PAN RNA virus to examine if the tailless PAN RNA mutants rescue the loss of virion production seen previously during the downregulation of KSHV PAN RNA. The results from these experiments would yield a deeper understanding of host-virus interactions and will provide insights into the importance of PABP-binding for the function of a nuclear noncoding RNA.</p><p>

Hepatitis B virus (HBV) is one of the world’s unconquered diseases, with 370 million chronically infected globally. HBV replicates in hepatocytes within the liver that exist under a range of oxygen tensions from 11% in the peri-portal area to 3% in the peri-central lobules. HBV transgenic mice show a zonal pattern of' viral antigen with expression in the peri-central areas supporting a hypothesis that low oxygen regulates HBV replication. We investigated this hypothesis using a recently developed in vitro model system that supports HBV replication. We demonstrate that low oxygen significantly increases covalently closed circular viral DNA (cccDNA), viral promoter activity and pre-genomic RNA (pgRNA) level, consistent with low oxygen boosting viral transcription. Hypoxia inducible factors (HIFs) regulate cellular responses to low oxygen and we investigated a role for HIF-1\(\alpha\) or HIF-2\(\alpha\) on viral transcription. A combination of HIF inhibitors and silencing of HIF-l\(\alpha\) and HIF-2\(\alpha\) ablated the effect of low oxygen on cccDNA and pgRNA, suggesting a role in regulating HBV transcription. This study highlights a new role for hepatic oxygen levels to regulate multiple steps in the HBV life cycle and this may impact on future treatments for viral associated pathologies.

<p> Current antiretroviral therapy (ART) is highly effective at blocking HIV-1 replication but does not cure the infection due to the persistence of latently-infected cells that are able to undergo cellular proliferation (1). The majority of HIV-1 proviruses that persist during ART are defective. Of the minority that are intact and replication competent, it is not known what fraction are transcriptionally active <i>in vivo</i> versus those that are transcriptionally silent (latently infected). To address this question, I determined the fraction of HIV-1 proviruses in populations of expanded cell clones that express unspliced, cell-associated RNA during ART in one individual. In total, 34 different cell clones carrying either intact or defective proviruses in &ldquo;Patient 1&rdquo; from Maldarelli, <i>et al.</i> (1) were assessed. We found that a median of 2.3% of cells within clones harboring replication-competent proviruses contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory T cell subset, including for the replication-competent AMBI-1 clone, which was the source of persistent viremia on ART. The fraction of cells within a clone that contained HIV-1 RNA was not different in clones with replication-competent vs. defective proviruses. However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia. These findings suggest that the vast majority of HIV-1 proviruses in persistently-infected cells, including replication-competent proviruses, are transcriptionally silent at any given time. This silence, if maintained over time, may allow infected cells to persist and expand during effective ART.</p><p>

The DNAs of human papillomavirus (HPV) types 4, 16 and 18 have been detected in biopsies of normal and malignant human oral mucosa by Southern blot hybridisation and polymerase chain reaction (PCR). By the former technique HPV-4, HPV-16 and HPV-18 DNAs were detected in three separate carcinomas, but only found in adjacent dysplastic and normal tissue by PCR. The PCR technique also allowed detection of HPV-16 and HPV-18 DNA in additional carcinomas and normal samples. The oral HPV-4 DNA has been molecularly cloned and extensive restriction analysis and nucleotide sequencing showed identity with the prototype HPV-4 DNA. The HPV-18 DNA detected by Southern blot hybridisation showed an altered restriction pattern in the El region of the viral genome; however direct nucleotide sequencing of PCR products from the E6 ORF showed no sequence alterations in either normal or malignant samples. HPV-16 DNA detected in one carcinoma by Southern blot hybridisation revealed altered PstI and Hpall restriction patterns as compared with the prototype viral genome. The expected 2.6kb Hpall and 1.55kb PstI bands, which overlap, were absent, and an additional band of reduced molecular weight was visible in the Hpall digest, suggesting that the oral HPV-16 genome had undergone a deletion or rearrangement. In a further two carcinoma samples positive for HPV-16 DNA by PCR, amplification of a late region fragment of the viral genome produced fragments of reduced molecular weight. When these PCR fragments were used as probes, hybridisation was observed to the 1.78kb PstI and 1.81kb Hpall-BamHI bands of HPV-16 DNA, and also (as a smear) to human genomic DNA from both tumour and normal samples. This suggests that the viral DNA in these samples had undergone recombination events with repetitive cellular sequences, perhaps as a prelude to viral integration or as a means of activating cellular genes. A keratinocyte culture (T45) derived from an oral squamous cell carcinoma was found to be non-tumorigenic in vivo. PCR analysis revealed that a proportion of cells in the culture contained HPV-16 early sequences. The establishment of HPV-positive and HPV-negative clones from this culture will provide an excellent system for studying the role of viral and cellular factors in oral squamous cell carcinogenesis.